Transport of organelles and macromolecular complexes through the cytoplasm is essential for every eukaryotic cell. This process is performed by motor proteins that use the energy of ATP hydrolysis to move many types of cargo along microtubules and actin filaments. The spatial and temporal control of motor-dependent transport is critical for cell division, organelle transport and positioning, and the movement of mRNA and protein complexes in the cytoplasm. Defects in organelle transport and motor-associated proteins contribute or cause many neurodegenerative diseases, and mistakes in motor- driven chromosome segregation can cause abnormal development and cancer. The goal of this proposal is to understand how movement of microtubule motors is regulated by accessory proteins and how movement along microtubules generates cell polarity. For analysis of proteins regulating the movement we will use cultured Drosophila S2 cells because they are highly sensitive to protein knock-down by RNAi and movement of cargo along microtubules is not perturbed by other cytoskeletal elements. For analysis of cell polarity we will use primary cultures of Drosophila neurons where microtubules and microtubule motors are key components required for growth of axons and dendrites. The specific aims of the proposal are: (i) To establish how transport is regulated by local signals (microtubule-binding proteins). (ii) To find contribution of proteins that are not associated with microtubules to transport regulation (iii) To use neuronal model to find how microtubules and microtubule-dependent transport generate formation of cell processes.